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. 2018 Dec 5;247(1):135–146. doi: 10.1002/path.5177

Figure 5.

PATH-5177-FIG-0005-c

ERK signaling promotes differentiation of LS174T goblet cells by inhibiting the NOTCH pathway. (A) Western blotting for the level of ERK phosphorylation (T202/Y204) and phosphorylation of ERK substrate FRA1 (S265) in LS174T cells treated for 3 h with inhibitors of either MEK (CI‐1040, 2 μm) or ERK (SCH772984, 1 μm). Total ERK2, FRA1 and β‐actin were used as loading controls. n = 3. (B) LS174T cells were treated with DMSO, 1 μm DBZ (γ‐secretase inhibitor), 2 μm CI‐1040 (MEK inhibitor) or 1 μm SCH772984 (ERK1/2 inhibitor) for 72 h before measuring MUC2 expression by RT‐qPCR. Expression was normalized to the reference transcripts PUM1, MRPL19 and RPL13A. n ≥ 5. (C) Alcian blue staining was performed on EtOH fixed cells following 72 h of treatment with DMSO, DBZ or CI‐1040. n = 3. (D) Western blotting to measure the level of HES1 expression and Notch cleavage (NICD) in LS174T cells treated with either DBZ or MEK/ERK inhibitors, with β‐actin used as a loading control. n = 3. (E) NOTCH1, NOTCH3 as well as DLL1 and DLL4 mRNA expression were measured by RT‐qPCR in LS174T cells treated with CI‐1040 and SCH772984. Expression was normalized to the reference transcripts PUM1, MRPL19 and RPL13A. n ≥ 9 for each treatment. Graphs present mean ± SEM. Student's t‐test *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.